Stabilized coenzyme test compositions

ABSTRACT

THERE ARE DISCLOSED STABILIZED COENZYME TEST COMPOSITIONS WHICH COMPRISE REDUCED NICOTINAMIDE-ADENINEDINUCLEOTIDE AND/OR REDUCED NICOTINAMIDE-ADENINE-DINUCLEOTIDE-PHOSPHATE IN ADMIXTURE WITH AT LEAST ONE SHGROUP-CONTAINING ORGANIC COMPOUND, SUCH AS FOR EXAMPLE, CYSTEIN, GLUTATHIONE, THIOGLYCOLLATES, N-ACETYL-HOMOOCYSTEIN ETC. THE COMPOSITIONS ARE POROUS POWDERS, SOLUBLE IN WATER. THEY NEED ONLY BE BROUGHT INTO SOLUTION AND ADDED TO THE MATERIAL TO BE INVESTIGATED FOR THE ANALYTICAL PROCEDURE TO BE CARRIED OUT. THERE ARE ALSO DISCLOSED THE METHODS OF MAKING AND USING THE COMPOSITIONS.

United States Patent 3,746,625 STABILIZED COENZYME TEST COMPOSITIONS Hans Ulrich Bergmeyer, Bahnhofstrasse 5a, Tutzing, Germany; Erich Bernt, Gruwalder Strasse 34, Munich, Germany; and Hans-Georg Rey, Hersfelderstrasse 18, Mannheim-Waldhof, Germany No Drawing. Continuation-impart of abandoned application Ser. No. 689,005, Dec. 8, 1967. This application Dec. 8, 1970, Ser. No. 96,243 Claims priority, application Germany, Dec. 12, 1966, B 90 26 942.1 Int. Cl. G01n 31/14 US. Cl. 195-103.5 R 14 Claims ABSTRACT OF THE DISCLOSURE This application is a continuation-in-part of Ser. No. 689,005, filed Dec. 8, 1967, and now abandoned.

The present invention relates to stabilized coenzyme test compositions and to the preparation thereof.

More particularly, the invention relates to stabilized coenzyme test compositions containing diand/or triphosphopyridine-nucleotide in reduced form, these two nucleotides also being known as reduced nicotinamideadenine-dinucleotide and nicotinamide-adenine-dinucleotide-phosphate as well as to a process for making and using such compositions.

In carrying out enzyme investigations, particularly as required for diagnostic purposes, it is of the greatest importance that the materials necessary for the enzyme reactions are available in a form ready for use and in the correct proportions. For instance, it is highly desirable to have available a coenzyme composition which is stabilized against deterioration and thus remains suitable for use over prolonged periods of time.

However, heretofore providing such coenzyme test preparations has met with great difi'iculties. Thus, it has been found that coenzymes which are relatively stable in pure form, are frequently rendered unstable by the presence of other components, e.g., substrates, other coenzymes, buffers, and the like, of the test preparations so that, in the course of storage, they suffer from such a loss of activity that the known coenzyme test preparations are of doubtful practical utility. This applies particularly to combined preparations which contain reduced nicotinamide-adenine-dinucleotide and/or reduced nicotinamideadenine-dinucleotide-phosphate.

In accordance with the invention it has surprisingly been found that SH-group-containing compounds have the ability to stabilize coenzyme test preparations.

Thus, according to the present invention, there are now provided stabilized, combined coenzyme test preparations 3,746,625 Patented July 17, 1973 which comprise reduced nicotinamideadenine-dinucleotide and/or reduced nicotinamide-adenine-dinucleotidephosphate, together with at least one SH-group-containing compound as stabilizer.

The SH-group-containing compounds which are suitable for use according to the present invention are preferably organic compounds, for example, glutathione, cysteine, cysteine carbamide, acetyl-cysteine, homocysteine thiolactone, N-acetyl-homocysteine thiolactone, thioglycollates or dithiothreitol, particularly in the case of combined coenzyme test compositions containing reduced nicotinamide-admine-dinucleotide.

It is, of course, to be understood that the stabilizers must be so chosen that they do not interfere with or exert any influence on the test reactions. This latter possibility can easily be ascertained by simple preliminary experiments and thereby avoided.

The SH-group-containing compounds which are particularly preferred are cysteine, cysteine carbamide and acetyl-cysteine. An especial advantage of these substances is that they are considerably cheaper than, for example, glutathione.

The new test compositions according to the present invention can be prepared by the following method:

(a) Admixing in water of substances necessary for carrying out the reaction such as reduced nicotinamideadenine-dinucleotide and/or reduced nicotinamide-adenine-dinucleotide-phosphate, possibly other coenzymes, as well as adjuvant substrates, buffer substances, other adjuvants and ossibly also fillers together with the SH-groupcontaining stabilizer, in the amounts necessary for carrying out the specific reaction to provide an aqueous solution, the pH value and volume of which can then, if necessary or if desired, be adjusted in the known manner;

(bl Filling this solution in the desired amounts into suitable containers; and

(c) Lyophilizing the contents of the containers and thereafter immediately closing the same.

As fillers, there may be used those non-ionic organic substances, such as gelatine, polyvinyl pyrrolidone, albumin and the like, which, as is known, simplify the lyophilization procedure.

The test compositions according to the present invention constitute porous powders which are readily soluble in water and which, when stored in sealed containers, retain their activity over a period of several years. The compositions of the invention represent a considerable advance in the field of reagent compositions for carrying out coenzyme studies and analyses. In accordance with the invention, it is now only necessary to dissolve the contents, comprising the instant compositions, of a sealed container, and such other materials, if any, needed for reaction, in a specified amount of water, add the material to be investigated, such as serum, and then to carry out a defined analysis, as for example, a determination of extinction values in a photometer.

In addition to stabilizer, reduced nicotinamide-adeninedinucleotide and/or reduced nicotinamide-adenine-dinucleotide-phosphate and the above-mentioned additional materials, the stabilized preparations according to the present invention can also contain one or more additional coenzymes.

The following examples are given for the purpose of illustrating the present invention and are in no way to be construed as limiting the invention. In each instance,

stabilized coenzyme test compositions are provided, i.e., compositions containing coenzymes which are stable for a comparatively long period of time in a lyophilized condition.

EXAMPLE 1 Determination of lactate dehydrogenase (LDH) Grams Na HP -2H O 2.43 NaH2P O4 0.21 Sodium pyruvate 0.012 Glutathione 0.30 Serum albumin 0.30 NADH 0.05

*NADH is reduced nicotinamide-adenine-dinucleotide.

were dissolved in 90 m1. bi-distilled water, and the pH value was adjusted to 7.6 with diluted solution of sodium hydroxide or hydrochloric acid. Then the solution was filled up to 100 ml. with bi-distilled water, frozen The above substances in the amounts indicated were worked up in the manner specified in Example 1.

EXAMPLE 3 Determination of the sorbitol dehydrogenase (SDH) Grams Triethanolamine hydrochloride 4.40 Sodium carbonate 0.50

Fructose 5.40 Glutathione 0.30 Serum albumin 0.30 NADH 0.05

The above substances in the amounts indicated were worked up in the manner specified in Example 1.

The stability of NADH with respect to temperature stress in the preparations in accordance with Examples 1, 2 and 3, above, compared to preparations without the stabilizing glutathione can be seen from Table I, below.

TABLE I.-STABILITY 0F NADH WITH AND WITHOUT GLUTATHIONE (GSH) AT 33 0.

[Values given in mg. 1NADH after redlssolution of the ophihzate] With GSH Without GSH Preparation of Example 1:

nitial value 50 50 1 wee 43 41 3 weeks 47 25 8 weeks 4.5 5 Preparation of Example 2:

Initial value 51 50 3 weeks 48 27 8 weeks 44 9 Preparation of Example 3- Initial va1ue 52 48 8 weeks 45 15 It will be understood that the specification and examples are illustrative but not limitative of the present invention and that other embodiments within the spirit and scope of the invention will suggest themselves to those skilled in the art.

What is claimed is:

1. A stabilized coenzyme test composition consisting of at least one member selected from the group consisting of reduced nicotinamide-adenine-dinucleotide and reduced nicotinamide-adenine-dinucleotide phosphate and at =least one SH-group containing compound as stabilizing agent wherein said SH-group containing compound is a member selected from the group consisting of cysteine, cysteine carbamide, acetyl-cysteine,. glutathione, homocysteine thiolactone, N-acetyl-homocysteine thiolactone, and thioglycollate.

2. A composition as claimed in claim 1 wherein said SH-group containing compound is cysteine carbamide.

3. A composition as claimed in claim 1 wherein said SH-group containing compound is acetyl-cysteine.

4. A composition as claimed in claim 1 wherein said SH-group containing compound is homocysteine thiolactone.

5. A composition as claimed in claim 1 wherein said SH-group containing compound is N-acetyl homocysteine thiolactone.

6. A composition as claimed in claim 1 wherein said SH-group containing compound is thioglycollate.

7. A stabilized coenzyme composition consisting of at least one coenzyme selected from the group consisting of reduced nicotinamide-adenine-dinucleotide and nicotinamide-adenine-dinucleotide-phosphate and as a stabilizer for said coenzyme stabilizingly efiective amounts of dithioerythritol.

8. A stabilized coenzyme composition consisting of at least one coenzyme selected from the group consisting of reduced nicotinamide-adenine-dinucleotide and nictotinamide-adenine-dinucleotide-phosphate and as a stabilizer for said coenzyme stabilizingly efiective amounts of cysteine.

9. A stabilized coenzyme composition consisting of at least one coenzyme selected from the group consisting of reduced nicotinamide-adenine-dinucleotide and nicotinamide-adenine-dinucleotide-phosphate and as a stabilizer for said coenzyme stabilizingly effective amounts of glutathione.

10. A stabilized coenzyme test composition consisting of:

Grams Na2HPO4 NaH PO -2H 0 a 0.21 Sodium pyruvate 0.012 Glutathione 0.30 Serum albumin 0.30 Reduced nicotinamide-adenine-dinucleotide 0.05

11. A stabilized coenzyme test composition consisting 12. A stabilized coenzyme test composition consisting of:

Grams Triethanolamine hydrochloride 4.40 Sodium carbonate 0.50 Fructose 5.40

Glutathione 0.30 Serum albumin 0.30 Reduced nicotinamide-adenine-dinucleotide 0.05

13. Process for stabilizing reduced nicotinamideadenine-dinucleotide or reduced nicotinamide-adeninedi uQkQtide-phosphate which consists of dissolving in water at least one member selected from the group consisting of reduced nicotinamide-adenine-dinucleotide and reduced nicotinamide-adenine-dinucleotide-phosphate and at least one SH-group containing a compound as stabilizing agent and thereafter lyophilizing the solution thereby formed, wherein said SH-group containing compound is a member selected from the group consisting of cysteine, cysteine carbamide, acetyl-cysteine, glutathione, homocysteine thiolacetone, N-acetylhomocysteine, thiolactone, and thioglycollate.

14. Process for carrying out a biological or chemical test procedure requiring reduced nicotinamide-adeninedinucleotide or reduced nicotinamide-adenine-dinucleotide phosphate as a coenzyme which comprises dissolving the composition of claim 1 in water, introducing into the solution thereby formed a liquid sample containing a material to be investigated and examining the resultant solution for evidence of the presence and/or amount of said material.

6 References Cited UNITED STATES PATENTS 3,413,198 11/1968 Deutsch 195--1O3.5R

OTHER REFERENCES ALVIN E. TANENHOLTZ, Primary Examiner 5 M. D. HENSLEY, Assistant Examiner US. Cl. X.R. 195--99; 252-402 

